Editorial Special Focus Issue: Bioanalysis of Large Molecules by LC–MS
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Validation of LC–MS/MS bioanalytical methods for protein therapeutics “
Of particular interest is LC–MS/MS analysis of therapeutic proteins via quantification of unique surrogate peptides that are produced by enzymatic digestion, a technique that is being widely used.
”
Keywords: enzymatic digestion • hybrid methods • PrD-LC–MS • protein digestion LC–MS/MS • quantitative protein LC–MS/MS • regulated biotherapeutic bioanalysis • validation
Background There are more than 100 protein and peptide drugs currently approved by the US FDA and now over 100 more are currently in active development by pharmaceutical companies worldwide [1] . The quantification of protein therapeutics has traditionally relied upon ligand-binding assays (LBAs). Such assays can be very sensitive and selective if properly developed using appropriate reagents, but are nevertheless susceptible to interferences due to circulating ligands, antidrug antibodies (ADAs), cross reactions and various forms of non-specific binding. LC– MS/MS analysis has emerged as an effective quantitative tool for protein bioanalysis. Of particular interest is LC–MS/MS analysis of therapeutic proteins via quantification of unique surrogate peptides that are produced by enzymatic digestion, a technique that is being widely used [1–4] . For simplicity, we will call this type of analysis protein digestion LC–MS/MS (PrD-LC–MS). PrD-LC– MS can be used as an alternative and supplemental approach to LBAs, and this has been extensively discussed in recent years [1–4] . Such LC–MS/MS methods, where the protein biotherapeutic is digested directly in the biomatrix prior to LC–MS/MS, can provide bioanalytical data with low interference from ADAs and other circulating ligands [5] . These methods can yield more representative quantification of total drug, but they may have limited sensitivity. Where higher sensitivity has been required, analyte enrichment or affinity capture concentration steps can be added either before [6] or after [7] pro-
10.4155/BIO.14.125 © 2014 Future Science Ltd
teolytic digestion. These enrichment methods can improve sensitivity by eliminating background signals either from the biomatrix itself or from the digest prior to peptide LC–MS/MS. As the methodology for PrD-LC–MS matures, there will be an increased need to use these assays in a regulated environment, both for good laboratory practice (GLP) toxicology studies and for regulated sample bioanalysis in clinical trials. Neither the recent EMA guidance [8] , nor the FDA draft Bioanalytical Method Validation (BMV) guidance [9] has specifically discussed the validation of PrD-LC–MS or its application to regulated bioanalysis. Several questions need to be addressed for any regulated PrD-LC–MS assay validation:
Faye Vazvaei‡ Roche Pharma Research & Early Development, Roche Innovation Center New York, Roche TCRC Inc., 430 East 29th St., New York, NY 10016, USA
• Which parameters of the assay must be addressed in validation? • How should each parameter be evaluated? • What are the overall acceptance criteria to be applied to such assays both for validation and for the acceptance of runs in routine sample analysis? The AAPS Consortium A consortium of members of the AAPS Bioanalytical Focus Group was formed under the auspices of its mass spectrometry protein bioanalysis subteam. Their purpose was to generate a White Paper reflecting the industry consensus on those parameters and the acceptance criteria that should be adopted as a starting point for validating
Bioanalysis (2014) 6(13), 1739–1742
Jeffrey X Duggan‡ Author for correspondence: Boehringer-Ingelheim Pharmaceuticals Inc., Bioanalysis & Metabolite Profiling, DMPK US, Ridgefield, CT 06877, USA Tel.: +1 203 791 6320; jeffrey.duggan@ boehringer-ingelheim.com ‡
Both authors contributed equally
part of
ISSN 1757-6180
1739
Editorial Vazvaei & Duggan PrD-LC–MS methods in the absence of official regulatory guidances for this purpose. The Consortium consists of scientists from numerous companies, representing both Pharma and CRO organizations with international presence. The scope of the White Paper is to specifically discuss the validation of quantitative determination of protein therapeutic molecules using a surrogate peptide produced by enzymatic digestion. The intent is for the paper to cover all the topics that are considered to be a part of the formal method validation process, with specific emphasis upon those procedures and practices that are unique to PrD-LC–MS methods. Consolidation of terminology In discussing PrD-LC–MS there are a number of terms that are used interchangeably in the literature and which can cause some confusion if not properly defined. An example of this is the surrogate peptide used for quantitation, a key component of the PrDLC–MS method. This peptide has been referred to as the “surrogate, target, signature, and quantitation” peptide in various publications and contexts. The White Paper proposes a glossary of terms that can be universally applied so as to improve clarity of PrD-LC–MS terminology. General acceptance criteria, precision & accuracy The validation criteria for acceptable precision and accuracy of PrD-LC–MS methods have not been specifically proposed in the literature or regulatory guidances; however, a recent review by Knutsson et al. makes the case that the higher complexity of PrDLC–MS methods, combined with a general lack of experience with them in the industry, suggests that the 4–6–20 (LBA) criteria be applied at this time [10] . The consortium of White Paper authors reached the same conclusion, but went on further to propose that both the demonstrated performance of the method in validation and the performance requirements may be used to drive the final acceptance criteria. Special considerations In developing and validating PrD-LC–MS methods, it should be kept in mind that these methods borrow elements from both small molecule chromatographic methods as well as ligand-binding methods. Practitioners with expertise in small molecules are new to this technology, and they should pay attention to potential problems unique to protein analytes, such as aggregation due to vigorous mixing. Other important considerations for PrD-LC–MS methods, especially those with immunocapture, include the behavior of the
1740
Bioanalysis (2014) 6(13)
molecule in the presence of soluble targets, circulating ligands, ADAs and other protein-specific interferents. The suitability and stability of critical reagents used in immunoaffinity enrichment steps will also come into play in this technique. On the other hand, scientists with a background solely in LBAs will have to add to their list of method validation experiments parameters such as extraction and digestion efficiency, as well as the measurement of matrix effects by calculation of the matrix factor. These PrD-LC–MS measurements require the synthesis of the surrogate peptide and the internal standard surrogate peptide. Finally, nonspecific binding of protein analytes to containers, or binding of peptides from the digest to columns and other components during analysis, can cause carryover or appear as instability. These, along with other special considerations, are discussed in detail in the following sections. Surrogate & monitoring peptides The surrogate peptide is unique to the analyte protein, and its chromatographic SRM LC–MS signal is used for quantification. The qualification of the surrogate peptide during method development and its characteristics for successful validation are discussed. Also discussed are the various types of internal standards that may be used in PrD-LC–MS methods, including stable labeled flanked and non-flanked versions of the surrogate peptide, and stable labeled versions of the intact protein drug molecule. Because PrD-LC–MS utilizes LC–MS/MS, the system can monitor multiple peptide SRMs during an analytical run. This permits the simultaneous analysis of additional peptide(s) derived from other unique regions of the molecule. These additional peptides, called monitoring peptides, may be used to derive additional information about the biotherapeutic which could be related to in vivo metabolism, especially for multi-subunit protein drugs that may be subject to cleavage in vivo. Enzymatic digestions & digestion efficiency The extent to which a proteolytic digestion treatment fully cleaves the analyte protein releasing a stoichiometrically related quantity of the surrogate peptide is defined by a PrD-LC–MS method’s digestion efficiency. Similar to the accepted concepts applied to recovery in small molecule validations, the digestion efficiency should be consistent across assay concentrations, and the percent efficiency should be high enough to achieve the desired assay sensitivity. The role of digestion efficiency and its definition in a validated PrD-LC–MS method is discussed in the White Paper.
future science group
Validation of LC–MS/MS bioanalytical methods for protein therapeutics
Standards & internal standards Peptide standards and internal standards must be specifically and carefully designed for each PrD-LC–MS assay. The standard material is the biotherapeutic protein molecule itself; and a stable labeled internal standard is recommended for each method, but the internal standards come in a variety of specific forms that depend upon the assay design. The internal standard may be a stable labeled version of the surrogate peptide, an extended version of the same peptide with small additional portions of the protein sequence added to the Cor N-termini ends of the peptide, or it may consist of the entire protein with isotopic stable label incorporation.
internal standard, and vice versa, also needs to be demonstrated. To determine selectivity for PrD-LC–MS assays, the criteria for small molecule chromatographic assays [8,11] should be followed (interference in blank matrix
For reprint orders, please contact [email protected]
Validation of LC–MS/MS bioanalytical methods for protein therapeutics “
Of particular interest is LC–MS/MS analysis of therapeutic proteins via quantification of unique surrogate peptides that are produced by enzymatic digestion, a technique that is being widely used.
”
Keywords: enzymatic digestion • hybrid methods • PrD-LC–MS • protein digestion LC–MS/MS • quantitative protein LC–MS/MS • regulated biotherapeutic bioanalysis • validation
Background There are more than 100 protein and peptide drugs currently approved by the US FDA and now over 100 more are currently in active development by pharmaceutical companies worldwide [1] . The quantification of protein therapeutics has traditionally relied upon ligand-binding assays (LBAs). Such assays can be very sensitive and selective if properly developed using appropriate reagents, but are nevertheless susceptible to interferences due to circulating ligands, antidrug antibodies (ADAs), cross reactions and various forms of non-specific binding. LC– MS/MS analysis has emerged as an effective quantitative tool for protein bioanalysis. Of particular interest is LC–MS/MS analysis of therapeutic proteins via quantification of unique surrogate peptides that are produced by enzymatic digestion, a technique that is being widely used [1–4] . For simplicity, we will call this type of analysis protein digestion LC–MS/MS (PrD-LC–MS). PrD-LC– MS can be used as an alternative and supplemental approach to LBAs, and this has been extensively discussed in recent years [1–4] . Such LC–MS/MS methods, where the protein biotherapeutic is digested directly in the biomatrix prior to LC–MS/MS, can provide bioanalytical data with low interference from ADAs and other circulating ligands [5] . These methods can yield more representative quantification of total drug, but they may have limited sensitivity. Where higher sensitivity has been required, analyte enrichment or affinity capture concentration steps can be added either before [6] or after [7] pro-
10.4155/BIO.14.125 © 2014 Future Science Ltd
teolytic digestion. These enrichment methods can improve sensitivity by eliminating background signals either from the biomatrix itself or from the digest prior to peptide LC–MS/MS. As the methodology for PrD-LC–MS matures, there will be an increased need to use these assays in a regulated environment, both for good laboratory practice (GLP) toxicology studies and for regulated sample bioanalysis in clinical trials. Neither the recent EMA guidance [8] , nor the FDA draft Bioanalytical Method Validation (BMV) guidance [9] has specifically discussed the validation of PrD-LC–MS or its application to regulated bioanalysis. Several questions need to be addressed for any regulated PrD-LC–MS assay validation:
Faye Vazvaei‡ Roche Pharma Research & Early Development, Roche Innovation Center New York, Roche TCRC Inc., 430 East 29th St., New York, NY 10016, USA
• Which parameters of the assay must be addressed in validation? • How should each parameter be evaluated? • What are the overall acceptance criteria to be applied to such assays both for validation and for the acceptance of runs in routine sample analysis? The AAPS Consortium A consortium of members of the AAPS Bioanalytical Focus Group was formed under the auspices of its mass spectrometry protein bioanalysis subteam. Their purpose was to generate a White Paper reflecting the industry consensus on those parameters and the acceptance criteria that should be adopted as a starting point for validating
Bioanalysis (2014) 6(13), 1739–1742
Jeffrey X Duggan‡ Author for correspondence: Boehringer-Ingelheim Pharmaceuticals Inc., Bioanalysis & Metabolite Profiling, DMPK US, Ridgefield, CT 06877, USA Tel.: +1 203 791 6320; jeffrey.duggan@ boehringer-ingelheim.com ‡
Both authors contributed equally
part of
ISSN 1757-6180
1739
Editorial Vazvaei & Duggan PrD-LC–MS methods in the absence of official regulatory guidances for this purpose. The Consortium consists of scientists from numerous companies, representing both Pharma and CRO organizations with international presence. The scope of the White Paper is to specifically discuss the validation of quantitative determination of protein therapeutic molecules using a surrogate peptide produced by enzymatic digestion. The intent is for the paper to cover all the topics that are considered to be a part of the formal method validation process, with specific emphasis upon those procedures and practices that are unique to PrD-LC–MS methods. Consolidation of terminology In discussing PrD-LC–MS there are a number of terms that are used interchangeably in the literature and which can cause some confusion if not properly defined. An example of this is the surrogate peptide used for quantitation, a key component of the PrDLC–MS method. This peptide has been referred to as the “surrogate, target, signature, and quantitation” peptide in various publications and contexts. The White Paper proposes a glossary of terms that can be universally applied so as to improve clarity of PrD-LC–MS terminology. General acceptance criteria, precision & accuracy The validation criteria for acceptable precision and accuracy of PrD-LC–MS methods have not been specifically proposed in the literature or regulatory guidances; however, a recent review by Knutsson et al. makes the case that the higher complexity of PrDLC–MS methods, combined with a general lack of experience with them in the industry, suggests that the 4–6–20 (LBA) criteria be applied at this time [10] . The consortium of White Paper authors reached the same conclusion, but went on further to propose that both the demonstrated performance of the method in validation and the performance requirements may be used to drive the final acceptance criteria. Special considerations In developing and validating PrD-LC–MS methods, it should be kept in mind that these methods borrow elements from both small molecule chromatographic methods as well as ligand-binding methods. Practitioners with expertise in small molecules are new to this technology, and they should pay attention to potential problems unique to protein analytes, such as aggregation due to vigorous mixing. Other important considerations for PrD-LC–MS methods, especially those with immunocapture, include the behavior of the
1740
Bioanalysis (2014) 6(13)
molecule in the presence of soluble targets, circulating ligands, ADAs and other protein-specific interferents. The suitability and stability of critical reagents used in immunoaffinity enrichment steps will also come into play in this technique. On the other hand, scientists with a background solely in LBAs will have to add to their list of method validation experiments parameters such as extraction and digestion efficiency, as well as the measurement of matrix effects by calculation of the matrix factor. These PrD-LC–MS measurements require the synthesis of the surrogate peptide and the internal standard surrogate peptide. Finally, nonspecific binding of protein analytes to containers, or binding of peptides from the digest to columns and other components during analysis, can cause carryover or appear as instability. These, along with other special considerations, are discussed in detail in the following sections. Surrogate & monitoring peptides The surrogate peptide is unique to the analyte protein, and its chromatographic SRM LC–MS signal is used for quantification. The qualification of the surrogate peptide during method development and its characteristics for successful validation are discussed. Also discussed are the various types of internal standards that may be used in PrD-LC–MS methods, including stable labeled flanked and non-flanked versions of the surrogate peptide, and stable labeled versions of the intact protein drug molecule. Because PrD-LC–MS utilizes LC–MS/MS, the system can monitor multiple peptide SRMs during an analytical run. This permits the simultaneous analysis of additional peptide(s) derived from other unique regions of the molecule. These additional peptides, called monitoring peptides, may be used to derive additional information about the biotherapeutic which could be related to in vivo metabolism, especially for multi-subunit protein drugs that may be subject to cleavage in vivo. Enzymatic digestions & digestion efficiency The extent to which a proteolytic digestion treatment fully cleaves the analyte protein releasing a stoichiometrically related quantity of the surrogate peptide is defined by a PrD-LC–MS method’s digestion efficiency. Similar to the accepted concepts applied to recovery in small molecule validations, the digestion efficiency should be consistent across assay concentrations, and the percent efficiency should be high enough to achieve the desired assay sensitivity. The role of digestion efficiency and its definition in a validated PrD-LC–MS method is discussed in the White Paper.
future science group
Validation of LC–MS/MS bioanalytical methods for protein therapeutics
Standards & internal standards Peptide standards and internal standards must be specifically and carefully designed for each PrD-LC–MS assay. The standard material is the biotherapeutic protein molecule itself; and a stable labeled internal standard is recommended for each method, but the internal standards come in a variety of specific forms that depend upon the assay design. The internal standard may be a stable labeled version of the surrogate peptide, an extended version of the same peptide with small additional portions of the protein sequence added to the Cor N-termini ends of the peptide, or it may consist of the entire protein with isotopic stable label incorporation.
internal standard, and vice versa, also needs to be demonstrated. To determine selectivity for PrD-LC–MS assays, the criteria for small molecule chromatographic assays [8,11] should be followed (interference in blank matrix
